Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to novel antigens and derivatives thereof derived from Borrelia burgdorferi, to methods of their production, to their use in human and animal medicine and diagnosis and to pharmaceutical compositions containing them.
• the present invention provides the cloning expression of a novel polymorphic B. burgdorferi lipoprotein, OspG. This has previously been known as lp77.
• OspG a novel polymorphic B. burgdorferi lipoprotein
• the deduced amino acid sequence of this protein exhibits no significant homologies to other known Borrelial antigens such as OspA, OspB, OspC, OspD, OspE, OspF and P27.
• These outer surface proteins exhibit between 41 % and 65% similarity with OspG (Table 1).
• Lyme disease is the most common vector borne infectious disease of the temperate climate.
• the etiological agent, the spirochete Borrelia burgdorferi causes a multisystemic illness in humans which may affect skin, nervous system, joints and heart (8, 44).
• B. burgdorferi strains isolated from different biological sources and geographic areas are heterogeneous (2, 19, 38, 45, 50) and it is assumed, that the patterns of disease manifestations are influenced by antigenic differences of the spirochetal strains.
• burgdorferi antigens such as outer surface protein A (OspA), OspB, pC and plOO, are employed for serological diagnosis and as putative candidates for vaccine development (13, 14, 36, 38, 40, 41).
• OspA outer surface protein A
• OspB OspB
• pC pC
• plOO outer surface protein A
• the present inventors have discovered a further lipoprotein from B. burgdorferi which is designated OspG.
• the mature protein is further characterised as having one large hydrophobic domain of about 20 amino acids at the amino- terminal portion of OspG.
• This N-terminal peptide corresponds to the leader signal peptide found in typical prokaryotic lipoprotein precursors.
• a cleavage site presumably recognized by a B. burgdorferi signal peptidase.
• OspG The potential cleavage site in OspG is found between serine at position 19 and cystein at position 20.
• the sequence around the cleavage site of OspG is L-1-l-S-C.
• the OspG gene is located naturally on the 55kb plasmid in B. burgdorferi ZS7. Weak cross reactivity of the OspG probe with the 45-kb plasmid was noted.
• OspG has the amino acid sequence substantially as set forth in figure 1. It is further characterised by being expressed only during infection and is not detectable in B. burgdorferi cultivated in vitro.
• the present invention provides an isolated protein derived from B. burgdorferi characterised in that it has a molecular weight of between 20-22kDa as determined by two-dimensional SDS gel electrophoresis and an isoelectric point of between 4.9 and 5.4.
• the present inventors further provide a protein or an immunologically or antigenically equivalent fragment or derivative thereof having at least 80% homology to the amino acid sequence depicted in figure 1.
• the protein and its corresponding DNA and RNA sequences find utility in both the vaccine and diagnostic field.
• the present invention provides a protein having at least 85% homology, more preferably 90% homology and most preferably at least 95% homology to the protein depicted in figure 1.
• the protein of the present invention may be a fusion protein, in which case the fusion protein is characterised by having a portion of its amino acid sequence or a fragment thereof. Preferably the portion is at least 80%, preferably 85%, more preferably 90%, and most preferably 95% homologous to the protein sequence of figure 1.
• the protein is at least 70% pure as determined by SDS polyacrylamide gel electrophoresis, and most preferably 80% pure, and more preferably at least 90% pure.
• the protein of the present invention maybe a lipoprotein or produced as a protein without any associated lipids.
• the lipoprotein When produced by recombinant techniques, the lipoprotein will be expressed with the signal sequence. Cleavage of the signal sequence, to remove the N-terminal 19 amino acids will result in a non-lipidated molecule being produced.
• Immunoblot analysis shows that OspG is recognised by sera from mice previously infected with intact spirochetes that suggests that the native protein is immunogenic in this species.
• the invention provides a DNA sequence encoding for a protein OspG or fragment or derivative thereof.
• the DNA sequence is substantially as set forth in Figure 1.
• the term substantially as used herein means at least 70% identity to the sequence set forth in Figure 1, preferably 80% identity and more preferably at least 85% and most preferably at least 95% identify.
• the present invention provides a DNA sequence having substantially the sequence depicted in Figure 1 , or a fragment thereof, or a DNA sequence which hybridises to said sequence and which codes for a protein which exhibits OspG antigenicity.
• the DNA of the present invention may be prepared by enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less.
• a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less.
• Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl2, 0.01M dithiothreito
• lmg/ml bovine serum albumin at a temperature of 4°C to ambient, generally in a volume of 50 ⁇ l or less.
• the chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982),or in other scientific publications, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C.
• the coding sequence can be derived from B. burgdorferi mRNA, using known techniques (e.g. reverse transcription of mRNA to generate a complementary cDNA strand), and commercially available cDNA kits.
• DNA polymers which encodes mutants of the protein of the invention may be prepared by site-directed mutagenesis of the cDNA which codes for the protein by conventional methods such as those described by G. Winter et al in Nature 1982, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6500, or deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter et al in Biochem. Soc. Trans., 1984, 12, 224-225.
• the present invention provides a process comprising the steps of: i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes said OspG protein or an immunogenic derivative thereof; ii) transforming a host cell with said vector; iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein; and iv) recovering said protein.
• 'transforming' is used herein to mean the introduction of foreign DNA into a host cell by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kingsman and A.J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988.
• the term 'transformed' or 'transformant' will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest.
• the expression vector is novel and also forms part of the invention.
• the replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the OspG protein, or fragments thereof, under ligating conditions.
• the DNA polymer may be preformed or formed during the construction of the vector, as desired.
• the choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses.
• the preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al cited above.
• the recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Mamatis et al cited above, or "DNA Cloning" Vol. II, D.M. Glover ed., IRL Press Ltd, 1985.
• a bacterial host such as E. coli may be treated with a solution of CaCl2 (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol.
• Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells.
• the invention also extends to a host cell transformed with a replicable expression vector of the invention.
• Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al and "DNA Cloning" cited above.
• the cell is supplied with nutrient and cultured at a temperature below 45°C.
• the product is recovered by conventional methods according to the host cell.
• the host cell is bacterial, such as E. coli it may be lysed, chemically or enzymatically and the protein product isolated from the resulting lysate or super variant.
• the product may generally be isolated from the nutrient medium or from cell free extracts.
• Conventional protein isolation techniques include selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.
• the expression may be carried out in insect cells using a suitable vector such as the Baculovirus.
• the protein is expressed in Lepidoptera cells to produce immunogenic polypeptides.
• a suitable vector such as the Baculovirus.
• the protein is expressed in Lepidoptera cells to produce immunogenic polypeptides.
• an expression cassette comprising the protein coding sequence, operatively linked to a baculovirus promoter, typically is placed into a shuttle vector.
• Such vector contains a sufficient amount of bacterial DNA to propagate the shuttle vector in E. coli or some other suitable prokaryotic host.
• Such shuttle vector also contains a sufficient amount of baculovirus DNA flanking the desired protein coding sequence so as to permit recombination between a wild-type baculovirus and the heterologous gene.
• the recombinant vector is then cotransfected into Lepidoptera cells with DNA from a wild-type baculovirus.
• the recombinant baculoviruses arising from homologous recombination are then selected and plaque purified by standard techniques. See Summers et al., TAES Bull (Texas Agricultural Experimental Station Bulletin) NR 1555, May, 1987.
• a process for expressing the CS protein in insect cells is described in detail in USSN 287,934 of SmithKline RIT (WO/US 89/05550). Production in insect cells can also be accomplished by infecting insect larvae.
• the protein can be produced in Heliothis virescens caterpillars by feeding the recombinant baculovirus of the invention along with traces of wild type baculovirus and then extracting the protein from the hemolymph after about two days. See, for example, Miller et al., PCT/WO88/02030.
• novel protein of the invention may also be expressed in yeast cells as described for the CS protein in EP-A-0 278 941.
• the present invention also relates to vaccine composition comprising OspG or fragment or derivative thereof.
• an aqueous solution of the protein(s) can be used directly.
• the protein, with or without prior lyophilization can be mixed or absorbed with any of the various known adjuvants.
• adjuvants include, but are not limited to, aluminium hydroxide, muramyl dipeptide and saponins such as Quil A.
• Particularly preferred adjuvants are, MPL (monophosphoryl lipid A) and 3D-MPL (3 De-O-acylated monophosphoryl lipid A).
• a further preferred adjuvant is known as QS21.
• 3 D-MPL can be obtained from Ribi Immunochem or by the methods disclosed in UK patent No.
• the proteins can be encapsulated within microparticles such as liposomes or associated with oil in water emulsions.
• the proteins can be conjugated to an immuostimulating macromolecule, such as killed Bordetella or a tetanus toxoid.
• the antigen of the present invention will contain other Borrelia antigens, in particular OspA.
• the proteins of the present invention may be expressed by live vectors such as BCG, Listeria or Salmonella and formulated as live vaccines using such vectors.
• Vaccine preparation is generally described in New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Maryland, 1978. Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, US Patent 4,372,945 and Armor et al., US Patent 4,474,757.
• the amount of the protein of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 ⁇ g of protein, preferably 1-200 ⁇ g. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects may receive an additional administration to enhance their immune response.
• the present invention also relates to antibodies, preferably monoclonal antibodies which are specific for OspG. Such antibodies fmd utility in the diagnosis and also in the prevention of Lyme disease.
• the invention provides a diagnostic kit comprising an OspG antigen.
• B. Burgdorferi strains used in this study were described elsewhere (45). Borrelia were grown in modified Barbour-Stoenner-Kelly II (BSK II) medium (2) at 33°C. Spirochetes were harvested by centifugation at 10,000 x g at 4°C for 20 min, washed two times in PBS, and enumerated by dark-field microscopy. Preparation and screening a B. burgdorferi expression library
• Genomic DNA was prepared from B. burgdorferi strain ZS7 by the lysozyme/SDS method, and DNA fragments were generated by sonication. Blunt- ended DNA was inserted into the pUEXl vector using an adaptor cloning strategy (7,31). The ligated DNA was transformed into E.coli MC1061 followed by expression screening using an immune sera taken from DBA/2 mice inoculated with 104 (and fewer) B.burgdorferi (ZS7) organisms.
• Total genomic DNA was extracted from Borrelia organisms as described previously (32). Approximately 5 ⁇ g of DNA was digested with 100 U of restriction nuclease (Hindlll) according to the manufacturer's recommendations (Boehringer, Mannheim). Samples were subjected to electrophoresis using a 0.7% agarose gel. DNA fragments were transferred to Hybond TM-N nylon membrane (Amersham) followed by UV-cross-linking and hybridization. Briefly, using 32p. labeled probes hybridization was done over night at 65°C in 0.5 M NaHPO4/7% NaDodSO ⁇ pH7.2.
• proteins were electroblotted for 1 hr at constant current (60 mA) onto Hybond C nitrocellulose sheets (Amersham) employing a semi-dry electroblotting chamber (BIO-RAD, Kunststoff, Germany) according to the manufacturers' recommendations.
• Blots were washed four times in the above mentioned buffer and twice in TBS and immunoreactive bands were then visualized by addition of 20 ml DEA-buffer (0.1M diethanolamine (Sigma), 0.02% NaN3, 5mM MgCl2, pH9.0] supplemented with 5-bromo-4-chloro-3- indolyphosphate (BCIP, Sigma; 165 ⁇ g/ml) and nitro blue tetrazolium (NBT, Sigma; 330 ⁇ g/ml) as substrate. The reaction was stopped by washing the membrane in 50mM Tris-HCl, 150 mM NaCl, 5 mM EDTA.
• DEA-buffer 0.1M diethanolamine (Sigma), 0.02% NaN3, 5mM MgCl2, pH9.0] supplemented with 5-bromo-4-chloro-3- indolyphosphate (BCIP, Sigma; 165 ⁇ g/ml) and nitro blue tetrazolium (NBT, Sigma;
• B. burgdorferi organisms were washed twice in PBS, transferred onto adhesion slides (Superior, Bad Mergentheim, FRG) (1 x 10-5 spirochetes/reaction field), fixed in absolute ethanol (2 min., -20°C) and air dried.
• the fixed spirochetes were incubated with the respective mAbs diluted spirochetes were incubated with a fluoresceine isothiocyanate moist chamber for 30 min. After three washings in PBS, the preparations were examined using a fluorescence microscope, and documented using a 400 ASA black and white film (HP5; Illford, UK).
• ELISA B.burgdorferi-specific antibodies were measured in a solid-phase ELISA system with B. burgdorferi B31 antigens as described previously (15).
• the ospG gene lacking the sequence encoding the hydrophobic leader peptide was PCR amplified with oligonucleotide primers 5'-
• GTGGATCCAAGATTGATGCGAGTAGTG-3' corresponds to nucleotides 61 to 79
• 5'-GTGAATTCTATTTTTTATCTTCTATATTTTGAGGCTCTG-3' corresponds to nucleotides 560 to 590.
• Plasmid pZS7 DNA was subjected to 30 cycles of PCR in a DNA Thermal Cycler (Bio-Med60). Denaturation was carried out at 94°C for 60 s, annealing at 48°C for 90 s and extension at 72°C for 90 s. The amplified fragment was ligated in frame with the glutathione S-transferase gene into the pGEX-2T vector after digestion with BamHI and EcoRI and used for transformation of DH5 ⁇ host cells.
• E. coli organisms transformed with plasmids carrying full-length (pZS77) of truncated (pOspG) versions of the ospG gene were grown in the presence of [9, 10- (n)- 3 H
• Immune sera were taken from mice previously either inoculated in the tail with 10 8 (C.B-17; IS anti-10 8 ) or 10 3 (DBA/2; IS anti-10 3 ) viable B. burgdorferi spirochetes of strain ZS7 or primed with 5-10 / xg of lipidated (lip) HpOspA (BALB/c; IS anti- lipOspA) or of recombinant (rec) recOspG (BALB/c; IS anti-recOspG) s.c. in adjuvant (ABM2; Sebac, Aidenbach, Germany) and boosted after 10 and 20 days.
• SCID mice were either left untreated or reconstituted with either normal mouse serum (NMS) or pooled Immune Sera (IS).
• NMS normal mouse serum
• IS pooled Immune Sera
• the amount of spirochete-specific IG (ELISA on whole B. burgdorferi cell lysates) transferred with the individual IS was as follows: IS anti-10 8 , 4,4 ⁇ g lg/mouse; IS anti-10 3 , 4,5 ⁇ g/mouse; IS anti- lipOspA, 5 ig/mouse; IS anti-recOspG, 72ng/mouse.
• the amount of specific lg was around 10-20 fold higher in IS anti-recOspG as compared to IS anti-10 3 .
• IS were given i.p.
• a B. burgdorferi ZS7 genomic DNA expression library was screened with an immune serum from mice previously infected with 10 3 spirochetes (IS anti-10 3 ).
• This IS was shown before to lack antibodies to OspA and OspB but convey protection in SCID mice against subsequent infection (35).
• this IS recognised four individual proteins with relative molecular masses of 19-20-kDa and two proteins of * 40-kDa when tested on immuno-blots from whole-cell lysates of strain ZS7 separated by two dimensional gel electrophoresis. Approximately 20 clones were identified with one clone, designated pZS77, being particular reactive.
• the recombinant plasmid pZS77 was subjected to restriction analysis, subcloning and sequencing.
• the nucleotide sequence of ospG together with the deduced amino acid sequence of OspG is shown in Figure 1.
• a consensus ribosome binding site (GGAG) is located 10 bp upstream of the ATG start codon of the ospG gene. Further upstream of this translational initiation sequence are the -10 region (TATATT), at positions -70 to -64, and the -35 region (TTGTTA), at positions - 105 to -100.
• TTATATT consensus ribosome binding site
• TTGTTA -35 region
• Two short inverted repeats with sequences ATATTT and TTACATTT were contained in this upstream region between positions -118 to -49.
• the ATG start codon of the ospG gene at position + 1 is followed by an open reading frame of 588 nucleotides, corresponding to a 196-amino-acid protein with a calculated molecular mass of 22.049 Da.
• a possible rho-independent terminator was identified between position 620 and 656. Alignment of the DNA sequence upstream of the ATG start codon of d e ospG gene with the recently reported promoter region of the ospE-ospF operon reveals identity of 94% as determined by the GAP algorithm.
• the ospG promoter contains two highly conserved octamer DNA motifs, ATGTATTT (at position -187 to -180) and AATTACAT (at position - 120 to -113), which have been shown before to be associated with protein binding sites for a negative regulator molecule, named MAT ⁇ 2 and to regulate gene expression during yeast differentiation (5, 25).
• ATGTATTT at position -187 to -180
• AATTACAT at position - 120 to -113
• lg immunoglobulin-like sequence ATTTGCAA (at position - 154 to -147) is located between both S. cerevisiae-like motifs that differs from the lg-octamer motif by only one transition substitution.
• the hydropathy profile of OspG suggest that the protein is largely hydrophilic with one hydrophobic domain of about 20 amino acids at the amino-terminal portion. This N-terminal region reveals similarities to leader signal peptides present in typical prokaryotic lipoproteins (51, 52). At the COOH-terminal end of the signal sequence is a putative signal peptidase 11 recognition motif Leu-X-Y-Z-Cys ( Figure 3a). The potential cleavage site in OspG is located between serine at position 19 and cystein at position 20. The calculated isolelectric point is at pi 5.2. Comparison of the amino acid sequence of OspG with the sequences of all known B.
• OspG exhibits the highest homology with OspF (65%) (Table 1). Furthermore, the basic N-terminal peptide motif M-N-K-K-M of OspG is identical to that observed for OspE and OspF.
• Plasmid and chromosomal DNA of several B burgdorferi strains were separated by pulse-field gel electrophoresis and hybridized to ospA and ospG specific probes.
• the ospA containing plasmid of strain ACA-1 was barely visible because of low amounts of DNA loaded onto the gel, but was seen after prolonged exposure (data not shown).
• Using the ospG probe a prominent band was seen with a linear plasmid of approximately 48 kb and a weaker one with a 45 kb plasmid of strain ZS7.
• Restriction fragment length polymorphism (RFLP) analysis of ospG with endonuclease Hindlll revealed at least seven distinct hybridization patterns among the 20 B. burgdorferi isolates tested: the majority of B. burgdorferi sensu stricto isolates are characterized by two hybridization fragments of 1.8 and 3.8 kb ( Figure 6, lane 1); 3 out of 6 B. garinii isolates tested did not hybridize with the ospG probe and B garinii strains 20047 and S90 exhibited fragments of 1.8 kb and 1.7 and 3 kb, respectively (data not shown); among strains of the species B.
• RFLP Restriction fragment length polymorphism
• afzelii at least three different hybridization patterns could be observed: one band of 2.4 kb for strain ACA-1 (lane 3) and two bands of either 1.9 and 2 kb for strain MMS (lane 4) or 2 and 5 kb for strain NE40 (lane 5).
• primers were selected in such a way that the final recombinant product lack the 20 amino acid residues composing the leader peptide (46).
• the amplified OspG-encoding product was inserted in frame with the carrier protein of the expression vector pGEX-2T (Pharmacia, Freiburg, Germany) and after induction with IPTG, and approximately 44-kDA GST-OspG fusion protein was obtained.
• the GST-OspG fusion protein was enriched from E. coli lysate by use of glutathione-agarose beads and subsequent digestion of the bound GST-OspG fusion proteins with a site-specific protease.
• Plasmid pZS77 encodes the full-length OspG precursor protein with its normal N-terminal signal sequence
• pOspG specifies a protein that has the first 21 residues of the OspG precursors replaced with the sequence Met-Lys.
• E coli cells containing the full-length ospG gene (plasmid pZS77) expressed a 20 kDa lipoprotein that partitioned into the detergent phase whereas lipoproteins could not be observed in DH5 ⁇ cells containing the truncated ospG gene.
• SCID mice were injected i.p. with either of the indicated IS and subsequently challenged with 10-5 B. burgdorferi organisms. The development of clinical arthritis and the presence of spirochetes in ear biopsies were monitored. Inoculated but otherwise untreated or (Normal Mouse Sera) NMS-treated SCID mice developed clinical arthritis starting from day 6 p.i. on with severe swellings of the tibiotarsal joints developing between days 13 to 24 (end point). As described before, SCID mice passively immunised with IS anti-10 8 or IS anti-10 3 and IS anti-lipOspA showed none or only marginal signs of clinical arthritis.
• pUEX a bacterial expression vector related to pEX with universal host specificity. Nucl. Acids Res. 15: 10056.
• a 9.0-kilobase-pair circular plasmid of Borrelia burgdorferi encodes an exported protein: Evidence for expression only during infection. Infect. Immun. 62:2653-2661.
• Met Asn Lys Lys Lys Met Lys Asn Leu lie lie Cys Ala Val Phe Val Leu 1 5 10 15
• MOLECULE TYPE DNA (genomic)
• ORGANISM Borrelia burgdorferi

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• 1995
  • 1995-08-11 WO PCT/EP1995/003213 patent/WO1996005313A1/en not_active Ceased
  • 1995-08-11 NZ NZ291818A patent/NZ291818A/en not_active IP Right Cessation
  • 1995-08-11 CN CN95195439A patent/CN1159831A/zh active Pending
  • 1995-08-11 JP JP8507024A patent/JPH10504455A/ja active Pending
  • 1995-08-11 BR BR9508802A patent/BR9508802A/pt not_active Application Discontinuation
  • 1995-08-11 PL PL95318672A patent/PL318672A1/xx unknown
  • 1995-08-11 AU AU33454/95A patent/AU687620B2/en not_active Ceased
  • 1995-08-11 CA CA002197712A patent/CA2197712A1/en not_active Abandoned
  • 1995-08-11 CZ CZ97424A patent/CZ42497A3/cs unknown
  • 1995-08-11 HU HU9701727A patent/HUT77251A/hu unknown
  • 1995-08-11 EP EP95929865A patent/EP0781338A1/en not_active Ceased
• 1997
  • 1997-02-17 KR KR1019970701019A patent/KR970705636A/ko not_active Withdrawn

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